Chromatofocusing of human hemoglobins: application to the quantitation of hemoglobin A2

Chromatofocusing of various hemoglobins was performed using standard ion exchangers and Ampholine solutions. With DEAE supports and a large difference between the adjusted pH of the ampholyte solution and the pH existing in the column, regular descending pH gradients were generated. Using CM-52 cellulose and small pH differences between the above, more complex pH profiles were obtained. Various hemoglobins generally eluted at a pH higher than their pI in the form of sharp peaks. The elution order was the same as with ion-exchange chromatographic procedures, including the same abnormalities with respect to the pI of some hemoglobins. Microchromatofocusing of DE-52 cellulose proved to be a sensitive and convenient technique for large-scale quantitation of HbA2.